Paraoxonase-1 (PON1) is a high-density lipoprotein (HDL)-associated serum enzyme thought to make a major contribution to the antioxidant and anti-inflammatory capacities of HDLs. However, the role of PON1 in the modulation of cholesterol efflux is poorly understood. The aim of our study was to investigate the involvement of PON1 in the regulation of cholesterol efflux, especially the mechanism by which it modulates HDL-mediated cholesterol transport. The enrichment of HDL(3) with human PON1 enhanced, in a dose-dependent manner, cholesterol efflux from THP-1 macrophage-like cells and ABCA1-enriched J774 macrophages. Moreover, an additive effect was observed when ABCA1-enriched J774 macrophages were incubated with both PON1 and apo-AI. Interestingly, PON1 alone was able to mediate cholesterol efflux from J774 macrophages and to upregulate ABCA1 expression on J774 macrophages. Immunofluorescence measurement showed an increase in PON1 levels in the cytoplasm of J774 macrophages overexpressing ABCA1. PON1 used an apo-AI-like mechanism to modulate cholesterol efflux from rapid and slow efflux pools derived from the lipid raft and nonraft domains of the plasma membrane, respectively. This was supported by the fact that ABCA1 protein was incrementally expressed by J774 macrophages within the first few hours of incubation with cholesterol-loaded J774 macrophages and that cyclodextrin significantly inhibited the capacity of PON1 to modulate cholesterol efflux from macrophages. This finding suggested that PON1 plays an important role in the antiatherogenic properties of HDLs and may exert its protective function outside the lipoprotein environment.